1. Field of Invention
The present invention relates to a series of compounds that inhibit tumors, being applied to tumor suppression.
2. Description of Related Art
A telomere is a region of repetitive DNA at the end of chromosome which protects the end of the chromosome from fusion and recombination with other chromosomes that may lead to structure change so as to keep chromosome stability and integrity. In the end of 1930s-1938. Muller, researchers that study genetic inheritance in fruit flies, found that chromosomes broken by external forces such as X-ray were unstable and had chromosomal rearrangements. In 1941, McClintock found genetic instability in hybrid corn with broken ends of chromosomes. Thus Muller said that in the end of the chromosome, there is a functional region without important genetic message and involved in chromosome stability and named it in Greek as Telomere (Telos : end ; meres ; part). The sequence of telomere was not found until 1978 when Blackburn found repeated sequence TTGGGG (SEQ ID NO: 1) in a flagellate called Tetrahymena. Later sequences of telomeres in other creatures were also provided. In eukaryote, repeated DNA sequence with a lot of Guanine is on two ends of the chromosome. As to human telomere, it has been shown to consists of a tandem array of 5-15 Kb of the simple repeat (TTAGGG)n (SEQ ID NO: 2) by Moyzis et al. in 1988. Moreover, there are at least two proteins-Telomeric repeat binding factors-TRF1, TRF2 bound with a T-loop that is formed by single strand of telomere DNA held together by telomere binding proteins. In fore-end of the T-loop, (both strands of the chromosome which are joined to an earlier point in the double-stranded DNA by the third stand end invading the strand pair to form a D-loop.)
the two strands of a double-stranded DNA molecule are separated for a stretch and held apart by a third strand of DNA that pairs with one of the strands so as to form a triple-stranded DNA called D-loop or displacement loop.
Generally, chromosomes DNA in eucaryotes is linear. In semiconservative replication, primase synthesizes a primer that is complementary of the end of a parent DNA. Then a DNA polymerase starts replication of a daughter strand. When the replication is over, the primer will have to be removed by another polymerase. An enzyme-ligase that joins DNA synthesizes new molecules to fill gaps in DNA. Yet a 5′ end of the daughter stand is unable to be filled so that an end replication problem is raised. Each DNA replication, end of DNA (telomere) loses 50-150 bp. Now a plurality of studies show striking correlation between the length of telomere and the times of cell division. When the length of telomere is shortened to a certain degree, cells become senescence or death. That means that telomeres loss may act as a mitotic clock. However, germ cells and tumor cells are different from normal somatic cells and their proliferative capabilities are unlimited. This means these cells must overcome senescence problems and have some mechanisms that maintain the length of telomere. Such mechanism is related to reactivation of telomerase.
The telomerase is a specific ribonucleoprotein reverase transcriptase inside human bodies that synthesize repeated sequence (TTAGGG)n (SEQ ID NO: 2) on the end of telomeres to extend the length of telomeres.
This enzyme consists of two subunits-telomerase RNA and telomerase catalytic subunit (hTERT). The hTR containing RNA includes repeated 11 nucleotides-5′-CUAACCCUAAC-3′ (SEQ ID NO: 3) that is complementary to TTAGGG (SEQ ID NO: 2) of telomere and is used as replication template to extend the length of telomere. As to the telomerase catalytic subunit, abbreviated as hTERT, it is a main catalytic subunit. Moreover, there are some other structure proteins such as TEP1, Dyskerin, p23, hsp90, L22 and hStau are used to stabilize the telomerase. Thus the telomerase uses RNA as template to produce a segment of DNA with the same sequence of telomere that connects with a 3′ end of the chromosome under the catalysis of the subunits of the reverase transcriptase so that the chromosomes will not be shortened progressively after replication and cells will continue to divide.
It is found from a plurality of studies that normal somatic cells express undetectable levels of telomerase while in highly proliferative cells such as germline cells, hematopoietic cells, trophoblast, endometric cells and over 85%˜90% tumor cells, telomerase is highly expressed. Thus expression of telomerase is an important characteristic of tumor cells. According to nature of tumors, it is found that in malignant tumors, high telomerase activity represents immortalization of tumor cells with unlimited proliferative capacity. On the contrary, most of benign tumors and normal tissues can't produce enough telomerase so that their proliferative capacity is limited. Since expression of telomerase is feature of tumors cells, telomerase has been considered as an important target in cancer diagnosis and therapeutics. Reacting with different sections in telomerase structure such as hTR formed by RNA, main catalytic subunit of telomerase hTERT, various telomerase inhibitors are studied to find out compounds that suppress unlimited proliferative activity of tumor cells given by telomerase.
Researches show that under normal physical situations and in the presence of K+Na+, a single strand on the end of chromosomes that is rich in guanine is capable of forming G-quadruplex structure. The G-quadruplex structure consists of a small TTA loops formed bh TTA segments and a guanine-tetrad formed by a square co-planar bonding of four guanine bases connected by hydrogen bondings. Once the interaction between the G-quadruplex with RNA (AATCCC) (SEQ ID NO: 4) of telomerase is inhibited for stabilizing G-quadruplex structure, the telomerase is unable to extend telomeres (add bases). Thus compounds that facilitate or stabilize formation of G-quadruplex structure in human telomeres may have potential in cancer treatment. In previous studies based on stabilization of G-quadruplex structure to develop compounds for suppression of telomere activity, the compounds are divided into two categories-tricyclic G-tetrad ligands and multi-cyclic G-tetrad ligands. 2,6-diamidoanthraquinone is a first tricyclic compound that was found to stabilize the G-quadruplex structure. This is due to delocalized skeletal structure of anthraquinone containing amido group that potentially enhances π-π stacking interactions with DNA bases. Thus nitrogen atoms on side chains interacts with guanine and the 2,6-diamidoanthraquinone plays an important role in the binding of deoxyribonucleotides. The tertiary nitrogen and the quaternary nitrogen are preferred because that an ionic interaction is generated between positive charges and bases. The anticancer activity of anthraquinone compounds depends on various functional groups on its two side chains. The functional groups on the end can bind to the minor grooves of DNA while the grooves beside a plane of the quadruplex structure are minor grooves. Thus different functional groups have great effects on binding capacity and sequence selectivity of the anthraquinone-DNA interaction.
Neidle and his co-workers compared a series of tricyclic Fluoreone derivatives with anthraquinone derivatives. Although the Fluoreone has lower cytotoxicity while on the G-Quadruplex stabilization, it's not as good as anthraquinone derivatives. That means the carbonyl groups on 9, 10-positions have positive effects on its stabilizing ability of G-quadruplex structure. Later, Neidle et al. synthesis a series of 3,6-bisamideacridines derivatives and expect that a nitrogen atom on the center of the chromophore can carry positive charge to stabilize the whole structure. Next 3,6,9-trisubstituted acridines such as BR-ACO-19 are developed and is shown to good abilities to stabilize the G-quadruplex structure. As to TMPyP4, PIPER, RHPS4 and a natural product-Telomestatine, they are all multi-cyclic molecules that stabilize G-quadruplex structure. These multi-cyclic compounds are easily intercalated in and overlapped with the G-quadruplex structure by their planar structure. Moreover, they all include nitrogen atoms with positive charge that enhances interactions between guanines and ions. Therefore, while developing and designing compounds that stabilize the G-quadruplex structure, the following factors should be considered: (a) a planar structure that favors intercalation of molecules within the G-quadruplex structure should be maintained. (b) it's optimal that the nitrogen atoms include positive charges around and the effect of the number of the charge on the interaction is 4+>3+>2+. (c) The compound activity may be affected by introduction hydrogen bonding and the length of side chains. (d) Addition of some certain functional groups into the compounds can enhance the interaction between the compounds and the G-quadruplex structure.
In 1925, Anthraquinone derivatives are used broadly on industries as dyes. A plurality of materials extracted from plants such as emodin, aloe-emodin include anthraquinone as bases and have tumor suppression effects. Furthermore, due to good anticancer effects of anthraquinone structure, the anthraquinone derivatives are synthesized and studied. For example, doxorubincin is a anticancer drug. Doxorubicin is well-known as a highly promising anticancer drug and is applied to a plurality of tumors such as leukemia with good therapeutic effects. It is supposed that anticancer mechanisms of Doxorubicin include intercalation into DNA, change of deoxyribonucleotide structure or function such as suppression of deoxyribonucleotide synthesis and formation of cleavable complex of DNA-topoisomerase II. Through formation of cleavable complex of DNA-topoisomerase II, the topoisomerase II activity is inhibited and free radicals that cause DNA cleavage are free radicals generated. These effects result in cell apoptosis. Yet doxorubincin has serious cardiac toxicity so that its clinical applications are restricted. As to mitoxantrone and ametantrone, they are clinically used anthraquinone derivatives with lower cardiac toxicity than doxorubicin. They are doxorubicin analogues developed in 1970s and their anticancer mechanism is supposed to be the same with doxorubicin-suppression of topoisomerase II activity. In clinical used, the two drugs are applied to leukemia, prostate cancer and multiple sclerosis treatment.
It is mentioned in previous papers that Amidoanthraquinone derivatives have anticancer effect in tumor cell test. In the beginning, Amidoanthraquinone is modified in 1,4-positions. Refer to the paper of Neidle in J. Med. Chem., 1988, compounds formed by substitution on a side chain of 1,4-diaminoanthraquinone can inhibit cancer cell line L1210. Anticancer drug-mitoxantrone used clinically is also a derivative of 1,4-diaminoanthraquinone. Thus the inventor starts from compounds substituted in 1,4-positions or other positions on side chains. In 2004, a series of 1,4-diaminoanthraquinone derivatives is revealed. In the study, methods of synthesizing 1,4-diamindoanthraquinone derivatives are disclosed. One of the ways is to dissolve 1,4-diaminoanthraquinone in N,N-diethylacetamide, then being connected with the substituents containing chloride by acylation (compound I2-4). The product is dissolved in ethanol and is added with substituents with amines, the compounds I5-9 are obtained through reflux of the solution. Another way is to dissolve 1,4-diaminoanthraquinone in N,N-diethylacetamide, various compounds I10-38 are obtained by various substituents connected after acylation (as shown FIG. 1).
In the paper, the mechanism of drug action of 1,4-diaminoanthraquinone derivatives is by binding to and stabilization of the G-quadruplex structure for further inhibition of telomerase. Then three cancer cell lines-C6 Cell (rat glioma cell), Hep G2 (human hepatocellular carcinoma cell line HepG2), 2.2.15 (HBV-producing human hepatoblastoma cell line) are used to evaluate cytotoxic effects of compounds I2-38 against cancer cell lines. Results of pharmacological tests show that compounds I5-7, I9, I28 and I31 have good cytotoxicity in C6 cell line, especially I9 and I28, twice as effective asadriamycin (IC50), yet still not as good as mitoxantrone. The structure formulas of compounds I5-7, I9, I28 and I31 are shown in FIG. 2. As to in vitro cytotoxic tests on Hep G2 cells, compounds I5, I6, I9 and I11 (as shown in FIG. 3) have higher cytotoxic effects. The effect of I11 is a bit higher than adriamycin while all four compounds are with higher activity than mitoxantrone. In cytotoxic tests of 2.2.15 cell line, compounds I5, I6 and I11 are most active and I6 has higher effect than adriamycin but not better than mitoxantrone. To sum up, compounds I5 and I6 have better cytotoxic effects on these tumor cell lines.
Due to tumor cell suppression of substituents at 1,4-position, the inventors further work on synthesis of 1,5-diaminoanthraquinone derivatives with different substituents at 1,5-positions. In 2006, a series of 1,5-diaminoanthraquinone derivatives is revealed. In the paper, methods of synthesizing 1,5-diamido-isomers are disclosed. One of the ways is to dissolve 1,5-diaminoanthraquinone in DMF and reflux heated, the being connected with the substituents containing chloride by acylation (compound II2-4). The product is dissolved in DMSO and is added with substituents with amines, the compounds II5-10 are obtained through reflux of the solution. Another way is to dissolve 1,5-diaminoanthraquinone in DMF, reflux heated, and connected with the substituents containing chloride by acylation. Then the product got is dissolved in DMSO, added with substituents with amines and reflux heated to get compounds II12-18. Or dissolve 1,5-diaminoanthraquinone in DMF and reflux heated, acylated to connect with substituents to obtain various compounds II19-27 (as shown FIG. 4).
As to mechanisms of drug action, it is mentioned above that 1,4-diaminoanthraquinone derivatives can bind to and stabilize the G-quadruplex structure so as to inhibit telomerase. No 1,5-diamido-isomers is found not only inhibit telomerase by above mechanim, another way to intercalate into DNA and inhibit topoisomerase II. Moreover, free radicals generated cause damage of DNA directly. In pharmacological tests, three cancer cell lines-C6 Cell (rat glioma cell), Hep G2 (human hepatocellular carcinoma cell line HepG2), 2.2.15 (HBV-producing human hepatoblastoma cell line) are used to determine cytotoxic effects of compounds II2-27 against cancer cell lines. Results of pharmacological tests show that compounds II5, II16 and II18 have better cytotoxic effect, compared to mitoxantrone. In cytotoxic tests on G2 Cells, the cytotoxic effect of compounds II5 and II18 on tumor cells are not bad, especially compound II18 is with higher cytotoxic effect on HBV-producing human hepatoblastoma than mitoxantrone. The chemical structures of mitoxantrone and compounds II5, II16 and II18 are shown in FIG. 5.
Next another series of compounds 2,6-diaminoanthraquinone derivatives are synthesized and compared with 1,4-diamido-isomers, 1,5-diamino-isomers, 1,5-diamido-isomers, 1,5-diacyloxy-isomers and 1,5-dithio-isomers to review their anti-tumor effects. A series of papers is published in 2007. In the papers, methods to synthesize 2,6-diamido-isomers is disclosed. One of the methods is to dissolve 2,6-diaminoanthraquinone in DMF and being connected with the substituents containing chloride by acylation (compound III31-32, III39).
Then the product got is dissolved in DMF, added with substituents with amines, the compounds III33-38 are obtained through reflux heated.
Another way is to dissolve 2,6-diaminoanthraquinone in DMF, being acylated to connect with various substituents for obtaining compounds III45-47 (as shown in FIG. 6).
As to pharmacological activity comparison between 1,5-diamido-isomers and 2,6-diamido-isomers, hTERT-BJ1 (human normal skin fibroblast cells) and H1299 (non-small cell lung cancer cells) cell lines are used.
The pharmacological tests used include TRAP assay that measures whether the compounds can stabilize telomeric G-quadruplex structure and SEAP assay that is used as the reporter system to evaluate activation and suppression of telomerase(PhTERT). The results of the TRAP assay show that compounds II5, II17, III33 and III34 (as shown in FIG. 7) can stabilize G-quadruplex and further suppress tolomerase activity with good effects. Moreover, it is noted that most of derivatives of 1,5-diacyloxy-isomers and 1,5-dithio-isomers has no effects of telomerase inhibition. As to previous synthesized 1,4-diamino1,5-diamino1,5-diamido2,6-diamido-isomers, once they are connected with substitutes like side chain substitution pattern of mitoxantrone, the compounds have stronger inhibition activity. In the SEAP assay, it is found that 1,5-diacyloxy-isomers1,5-dithio-isomers1,4-diamido-isomers can activate hTERT-BJ1 cells yet 1,5-diamido-isomers derivatives and 2,6-diamido-isomers derivatives have no effects on hTERT-BJ1 cells or H1299 cells.
Until now, anthraquinone derivatives with substituents at various positions have been synthesized. The derivatives substituted at positions of 1,4, 1,5, 2,6, or 2,7 are all found with telomerase inhibition activity in pharmacological tests, especially derivatives with amido substituents in the side chain, the inhibition of telomerase activity is obvious.
In order to overcome shortcomings of traditional anticancer drug-doxorubicin and base on synthesis anthraquinone derivatives with substituents at different positions for tumor inhibitions, there is a need to provide anti-cancer compounds and manufacturing methods thereof. The anticancer compounds are a series of 1,8-diamidoanthraquinone derivatives with amino compounds having a novel chemical structure yet without side effects such as serious cardiac toxicity of traditional anticancer drugs-doxorubicin.